This invention is directed to novel nucleic acid probes that can be used with stringent hybridization conditions thereby decreasing background readings and improving the ease and accuracy of probe use.
In recombinant DNA technology and related fields such as diagnostics it is necessary to possess means for detecting specific nucleic acid sequences. The basic building blocks from which nucleic acids are constructed are nucleotides. The major nucleotides found in living cells are adenosine, guanosine, cytidine, thymidine and uridine. Nucleic acids contain the information for the transfer of genetic information from one generation to the next, as well as for the expression of this information through protein synthesis. Therefore, genetic materials can be evaluated and manipulated on the basis of nucleotide sequences. Based on this understanding of genetic makeup, probes can be designed to perform a variety of functions in the field of recombinant DNA technology and related areas.
Probes are oligomeric or polymeric nucleic acid molecules with complementary sequences to a nucleic acid sequence of interest. Through the use of probes, the ability to identify, localize, and detect nucleic acid sequences can be readily accomplished. Probes are not only useful in laboratory techniques and diagnostics, but are also useful in the field of therapeutics.
Typically, probes are used in determining the presence of a specific nucleic acid sequence. This involves digesting DNA or RNA with restriction endonucleases to produce smaller fragments. The fragments are separated by molecular weight and transferred and bound to a filter. The filter is then incubated with labelled probes consisting of single strands of DNA or RNA with nucleotide sequences complementary to a specific region in the DNA or RNA that is being detected. If the labelled probe binds to one of the nucleic acid bands on the filter, the sequence of interest resides in that band. Thus, probes may be used to determine the presence of specific genes, pathogens, human and non-human DNA or RNA, natural or foreign DNA or RNA and a variety of other detectable compositions.
Whether a hybrid nucleic acid molecule forms accurately between a probe and target sequence depends on two important factors. The probe should bind only to the desired target sequence, and the hybrid nucleic acid molecule thus formed should be correctly and sensitively identified. Therefore, it is of great advantage to have a probe that is both capable of recognizing and hybridizing to only its complement and is also capable of being accurately and sensitively identified.
The use of probes in determining the presence of specific genomic regions has been possible for some time, see Southern, J. Mol. Biol., 98:503, 1975. Albarella et al., U.S. Pat. No. 4,563,417, describe hybridization which occurs between sample nucleic acid and the probe in nucleic acid hybridization assays detected by an antibody that binds to intercalation complexes formed in association with hybridized probe. The use of probes containing psoralen derivatives that form covalent bonds with the target DNA when photochemically activated has been described in Yabusaki et al., U.S. Pat. No. 4,599,303. Specific DNA probes in diagnostic microbiology have been disclosed in Falkow et al., U.S. Pat. No. 4,358,535. There are a variety of probes available for detection of a variety of nucleic acid sequences. Despite the variety of probe strategies available, it would greatly improve probe use to have a probe capable of withstanding stringent hybridization conditions to eliminate background readings and to improve the ease, accuracy and sensitivity of probe use.